Liquid-changing culture medium and application thereof

A medium and basal medium technology, applied in the culture process, tissue culture, cell culture active agent, etc., can solve the problems of low BSA residue production process, high cost of lentivirus, and long cycle of cell acclimation.

Pending Publication Date: 2022-03-22
GUANGZHOU FINELMMUNE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of a single production of lentivirus is high, and the cycle of cell adaptation is long, so it is not suitable for clinical projects with less demand for GMP-grade lentivirus
[0006] Therefore, there is still a lack of a medium suitable for the production of lentivirus by serum-free adherent 293T cells, and a production process for obtaining high titers and low BSA residues

Method used

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  • Liquid-changing culture medium and application thereof
  • Liquid-changing culture medium and application thereof
  • Liquid-changing culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A method for packaging lentivirus of serum-free adherent 293T cells, comprising the following steps:

[0036] S1: HEK293T cells (ATCC, Cat. No.: 11268) do not need to be acclimatized to low serum levels. After resuscitation and culture for two generations, lentivirus packaging is carried out. One day before packaging, 2.2×10 7 Cells were inoculated into T175 culture flasks filled with 10% FBS+DMEM, and the cell density must reach 90% the next day;

[0037] S2: Take out the cells to be packaged and replace with packaging medium (the basic medium is: DMEM, and add 5% FBS, 5% serum replacement);

[0038] S3: The calcium phosphate method is used for plasmid transfection. The expression plasmid is about 9000kb in size and has a GFP fluorescent label. The expression of the plasmid is as follows figure 1 shown. ;

[0039]S4: Plasmid transfection 4-6h replaces serum-free liquid exchange medium, the liquid exchange medium includes basal medium and shape maintenance agent, and...

Embodiment 2

[0044] A method for packaging lentivirus of serum-free adherent 293T cells, comprising the following steps:

[0045] S1: HEK293T cells do not need to be acclimatized with low serum. After two generations of resuscitation culture, lentivirus packaging is carried out. One day before packaging, 2.2×10 7 Cells were inoculated into T175 culture flasks filled with 10% FBS+DMEM, and the cell density must reach 90% the next day;

[0046] S2: Take out the cells to be packaged, and replace the packaging medium (the basic medium is: DMEM, and add 5% FBS, 5% serum replacement);

[0047] S3: The calcium phosphate method is used for plasmid transfection, and the expression plasmid size is about 9000kb;

[0048] S4: Plasmid transfection 4-6H replaces the serum-free liquid-exchange medium, and the liquid-exchange medium comprises basal medium and shape maintenance agent, and wherein basal medium is DMEM medium (gibco, product number C11995), and the composition of shape maintenance agent inc...

Embodiment 3

[0053] A method for packaging lentivirus of serum-free adherent 293T cells, comprising the following steps:

[0054] S1: HEK293T cells do not need to be acclimatized with low serum. After two generations of resuscitation culture, lentivirus packaging is carried out. One day before packaging, 2.2×10 7 Cells were inoculated into T175 culture flasks filled with 10% FBS+DMEM, and the cell density must reach 90% the next day;

[0055] S2: Take out the cells to be packaged, and replace the packaging medium (the basic medium is: DMEM, and add 5% FBS, 5% serum replacement);

[0056] S3: The calcium phosphate method is used for plasmid transfection, and the expression plasmid size is about 9000kb;

[0057] S4: Plasmid transfection 4-6H replaces the serum-free liquid-exchange medium, and the liquid-exchange medium comprises basal medium and shape maintenance agent, and wherein basal medium is DMEM medium (gibco, product number C11995), and the composition of shape maintenance agent inc...

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Abstract

The invention relates to a liquid-changing culture medium and application thereof, and belongs to the technical field of virus vector packaging. The liquid-changing culture medium comprises a basic culture medium and a form maintaining agent, wherein the form maintaining agent is prepared from the following components in percentage by volume: 3 to 10 percent of serum substitute, 1 + / -0.1 g / L of insulin, 0.55 + / -0.1 g / L of transferrin, 0.00067 + / -0.0001 percent of sodium selenite, 0.20 + / -0.05 g / L of ethanolamine, 1 + / -0.02 mmol / L of sodium pyruvate and 25 + / -5 mu mol / L of chloroquine phosphate. The form maintenance agent is added into the basic culture medium, so that the good cell state is maintained through the supplement of nutrient substances, and the lentivirus with higher titer than the lentivirus packaged by 10% fetal calf serum can be obtained by matching with a small amount of serum substitute (serum substitute).

Description

technical field [0001] The invention relates to the technical field of viral vector packaging, in particular to a liquid-exchanging medium and its application. Background technique [0002] Lentiviral vector is a gene therapy vector capable of infecting both dividing cells and non-dividing cells, and is commonly used in Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T). [0003] At present, the production of lentivirus is mainly divided into two process systems, one is the adherent system using cell factories with 1-40 layers, and the other is the suspension system using fermentation culture technology after 293T cells have been acclimatized without serum. The virus attachment process often uses low serum to acclimatize the cells and then package the lentivirus, which has a long acclimation cycle and still has the problem of low serum in the packaging process. Secondly, there are large batch-to-batch differences in fetal bovine serum, which has a certain impact on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0686C12N15/85C12N2501/33C12N2500/24C12N2500/30C12N2500/46C12N2500/36C12N2501/999C12N2501/998C12N2501/10C12N2500/34C12N2740/15052C12N2510/00C12N2800/107
Inventor 卞超超刘海平李翔张雁陈自创谢秉霖
Owner GUANGZHOU FINELMMUNE BIOTECHNOLOGY CO LTD
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